EP2654801B1 - Ciblage de cellules souches de gliome par une inhibition fonctionnelle spécifique de l'oncogène mir-138 pro-survie - Google Patents

Ciblage de cellules souches de gliome par une inhibition fonctionnelle spécifique de l'oncogène mir-138 pro-survie Download PDF

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EP2654801B1
EP2654801B1 EP11849941.7A EP11849941A EP2654801B1 EP 2654801 B1 EP2654801 B1 EP 2654801B1 EP 11849941 A EP11849941 A EP 11849941A EP 2654801 B1 EP2654801 B1 EP 2654801B1
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oligonucleotide
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glioma
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Prabha SAMPATH
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Definitions

  • MicroRNAs are endogenous small noncoding RNAs that regulate various physiological pathways such as cellular differentiation and proliferation via gene silencing. More specifically, they bind to complementary sequences in target mRNAs and suppress target gene expression.
  • miRNAs Alterations in miRNA expression are known to play a critical role in cancer initiation, progression and metastasis (Calin and Croce, 2006; Ma et al., 2007). The robust connection between miRNAs and tumorigenesis is emphasized by the differential expression profiles of miRNAs from healthy tissue across cancers (Ambros, 2004; Bagga and Pasquinelli, 2006; Bartel, 2004). Several miRNAs have been found to be aberrantly expressed in solid tumors as determined by high throughput microarray techniques. In addition, recent evidence indicates that miRNAs might also function as tumor suppressors or activators.
  • Examples include miR-127, and miR-15a / miR-16-1, which target BCL6 and BCL2 anti-apoptotic genes, respectively, and the let-7 family members, which target the Ras oncogenes (Esquela-Kerscher and Slack, 2006; Slack and Weidhaas, 2006; WO2008095096 ). Further, impaired miRNA regulatory network is known to be one of the key mechanisms in brain tumor pathogenesis (Bottoni et al., 2005; Chan et al., 2005).
  • Malignant gliomas account for approximately 70% of the 22,500 new cases of malignant primary brain tumors that are diagnosed in adults in the United States each year.
  • Malignant gliomas comprise of grade-III anaplastic astrocytoma and grade-IV glioblastoma multiforme (GBM) lesions that are highly invasive and display histological evidence of malignancy.
  • GBM is a lethal intracranial malignancy with a median survival of less than 12 months (Legler et al., 1999).
  • Composed of a heterogeneous mixture of poorly differentiated neoplastic astrocytes malignant gliomas primarily affect adults, and preferentially occur in the cerebral hemispheres. Treatment of malignant gliomas is palliative and includes surgery, radiotherapy, and chemotherapy and frequently recur after radiation as focal masses (Garden et al., 1991).
  • tumor-initiating cells or cancer stem cells
  • brain tumors Galli et al., 2004; Hemmati et al., 2003; Singh et al., 2004.
  • This cellular fraction of the tumors is capable of initiating tumors similar to the parental tumor when transplanted into a secondary site (Reya et al., 2001).
  • GSCs glioma stem cells
  • NSCs normal neural stem cells
  • GSCs also possess the self-renewal potential.
  • GSCs have the potential to recapitulate original polyclonal tumors when xeno-grafted to nude mice. They are chemo-resistant and radiation-resistant and therefore responsible for tumor progression and recurrence after conventional therapy.
  • Altered expression (e.g., reduced/loss-of expression) of a miRNA may lead to aberrant expression of its target proteins, resulting in altered phenotype.
  • over-expression of miR-34a inhibits proliferation, disrupts tumorsphere formation, or induces differentiation of GSCs, but fails to eliminate GSCs (Guessous et al., 2010; Li et al., 2009).
  • the present invention is based on unexpected discoveries that (i) the levels of microRNA-138 (miR-138) increase in glioma stem cells (GSCs) isolated from MG patients and in GBM patient tumor samples (primary and secondary) as compared to those in normal neural stem cells (NSCs) and in healthy controls, and (ii) knockdown of miR-138 results in suppression of cell proliferation, induction of apoptosis, and reduction of tumorigenicity.
  • GSCs glioma stem cells isolated from MG patients and in GBM patient tumor samples (primary and secondary) as compared to those in normal neural stem cells (NSCs) and in healthy controls
  • knockdown of miR-138 results in suppression of cell proliferation, induction of apoptosis, and reduction of tumorigenicity.
  • one aspect of this disclosure relates to a method for treating malignant gliomas, including administering to a subject in need thereof an effective amount of an oligonucleotide (an antagomir) specifically targeting miR-138, which can be a hairpin oligonucleotide.
  • the oligonucleotide can comprise a core sequence having the nucleotide sequence of 5'-CGGCCTGATTCACAACACCAGCT-3' (SEQ ID NO:1), which targets the sequence of human mature miR-138.
  • the oligonucleotide has the nucleotide sequence of 5'-GAGCTGGTATTGTGAATCAAGCAGCTTCCTGTCAGCGGCCTGATTC ACAACACCAGCTTTTTT-3' (SEQ ID NO:2).
  • one or more of the nucleotides in the oligonucleotide are modified by, e.g., a 2'-O-methoxyethyl group, a 2'-O-methoxy group, or a phosphorothioate group.
  • the present disclosure provides a method for inhibiting GSC cell growth, including contacting GSCs with an effective amount of any of the oligonucleotides described above.
  • the contacting step can be performed by administering the oligonucleotide to a subject in need of the treatment.
  • this disclosure provides a method for prolonging survival in malignant glioma patients (e.g., human patients) with an effective amount of any of the above-described oligonucleotides.
  • the patients subjected to this method have undergone or are in the course of conventional therapy, including surgery, radiotherapy, and/or chemotherapy.
  • oligonucleotide administered to such a subject can be in an amount sufficient to suppress GSC proliferation or in an amount sufficient to induce GSC apoptosis in the subject.
  • a pharmaceutical composition for use in treating MG, prolonging survival of a MG patient, or inhibiting GSC growth the pharmaceutical composition containing any one or more of the oligonucleotides described herein and a pharmaceutically acceptable carrier; and (b) the use of the just-described pharmaceutical composition in manufacturing a medicament for any of these purposes.
  • the present disclosure provides methods for monitoring MG progress, methods for assessing efficacy of a MG treatment, and methods for identifying GSCs, all relying on the level of miR-138 as a biomarker.
  • a method for monitoring MG progress includes at least the following steps: obtaining samples containing tumor cells from a patient (e.g., a human patient or a laboratory animal) suffering from MG at multiple time points, quantifying the levels of miR-138 in the samples, and assessing whether MG progresses in the subject. An increase in miR-138 levels over time indicates progress of MG.
  • the quantifying step can be performed by any conventional method, including, e.g., quantitative real-time PCR or in-situ hybridization.
  • a method for assessing efficacy of a MG treatment in a patient includes obtaining samples containing tumor cells from the patient before, during, and/or after the treatment, quantifying levels of miR-138 in the samples via a conventional method (e.g., those listed above), and assessing efficacy of the treatment based on the level of miR-138. No change or a decrease in the levels of miR-138 after the treatment or along the course of the treatment indicates that the treatment is effective.
  • the samples are from the patient before and after the treatment and no change or a decrease in the level of miR-138 after the treatment indicates that the treatment is effective.
  • the samples are from the patient at multiple intervals during the treatment and no change or a decrease in the levels of miR-138 along the course of the treatment indicates that the treatment is effective.
  • a method for identifying GSCs includes quantifying the level of miR-138 in a sample suspected of containing GSCs via a conventional method as those described herein, and determining whether the sample contains glioma stem cells based on the level of miR-138.
  • An elevated miR-138 level in the sample as compared to a predetermined standard e.g., the level of miR-138 in non-tumor cells such as normal neural stem cells
  • the sample suspected of containing GSCs can be obtained from a human subject.
  • this method further comprises a step of assessing whether the human subject is suspected of having or at risk for malignant glioma.
  • Presence of GSCs in the sample indicates that the human subject is suspected of having or at risk for malignant glioma.
  • the method further comprises assessing the risk of tumor progression or tumor metastasis in a malignant glioma patient. Presence of GSCs in the sample is indicative of an increased risk for malignant glioma progression or metastasis.
  • MG malignant glioma
  • miR-138 is a well-characterized endogenous microRNA found in many species, including human and mouse. Information regarding miR-138 can be obtained from the microRNA database (www.mirbase.org) or from GenBank. Two putative precursors, miR-138-1 (GenBank Accession No. MI0000476) and pre-miR-138-2 (GenBank Accession No. MI0000455), have been identified, which are encoded on human chromosomes 3 and 16 and are 99 and 84 nucleotides in length, respectively (Griffiths-Jones, 2004; Lagos-Quintana et al., 2002; Weber, 2005).
  • nucleotide sequences of Pre-miR-138-1, Pre-miR-138-2, and mature miR-138 are provided below:
  • an effective amount of an oligonucleotide that targets miR-138 can be for use in treating MG in a subject in need of the treatment.
  • treating refers to the application or administration of a composition including one or more active agents to a subject, who has MG, a symptom of the disease, or a predisposition toward the disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of the disease, or the predisposition toward the disease.
  • an effective amount of the oligonucleotide noted above is for use in inhibiting glioma stem cell (GSC) growth, either in vitro or in a subject that needs the treatment. Inhibition of GSC growth reduces the risk for malignant glioma development, progression, and/or metastasis.
  • GSC glioma stem cell
  • an effective amount of a miR-138-targeting oligonucleotide is for prolonging survival of malignant glioma patients.
  • the malignant glioma patients have undergone, are in the course of, or will be subjected to conventional therapy, such as surgery, radiotherapy, or chemotherapy.
  • a malignant glioma patient subjected to this method is expected to have increased survival prospect as compared to malignant glioma patients with matched factors that may affect survival prospect (e.g., disease severity, age, gender, lifestyle, and conditions unrelated to malignant glioma) and not treated by this method.
  • an effective amount refers to the amount of an miR-138-targeting oligonucleotide that alone, or together with further doses or one or more other active agents, produces the desired response, e.g., inhibiting GSC growth via suppressing GSC proliferation and/or inducing GSC apoptosis, or prolonging survival of a malignant glioma patient.
  • the desired response may be inhibiting the progression of tumor growth. This may involve only slowing the progression of MG temporarily, although more preferably, it involves halting the progression of the tumor permanently and/or reducing the volume of the tumor.
  • the desired response to treatment of MG also can be delaying the onset or even preventing the onset of the tumor, or delaying metastasis or even preventing metastasis of the tumor.
  • Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight; the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • Body surface area may be approximately determined from height and weight of the patient.
  • a subject in need of any of the above-described treatments can be a subject (e.g., a human) suffering from MG (e.g., anaplastic astrocytoma or glioblastoma multiforme), suspected of having MG, or at risk for developing MG.
  • MG e.g., anaplastic astrocytoma or glioblastoma multiforme
  • a subject having MG can be identified via a routine medical procedures, including, but are not limited to, physical examination, pathological analysis, computed tomography (CT), and magnetic resonance imaging (MRI).
  • CT computed tomography
  • MRI magnetic resonance imaging
  • a subject suspected of having MG may show one or more symptoms of the disease, including seizure, nausea and vomiting, headache, hemiparesis, and a progressive memory, personality, or neurological deficit due to temporal and frontal lobe involvement.
  • Such subjects can be identified via routine medical procedures.
  • a subject at risk for developing MG possesses one or more risk
  • An oligonucleotide that targets miR-138 is a nucleic acid molecule (either DNA or RNA), at least a portion of which is complementary (i.e., completely or partially) to a fragment of miR-138, i.e., capable of forming a double-strand duplex with miR-138 via base-pairing according to the standard Watson-Crick complementarity rules.
  • Such an oligonucleotide is capable of reducing the level of endogenous miR-138 or inhibiting miR-138 activity.
  • An miR-138-targeting oligonucleotide preferably having a maximum length of 100 nucleotides (nts), can be a double-stranded molecule or a hairpin molecule including a 21-23nt sense sequence (complementary to a fragment of miR-138), a short linker, an antisense sequence, and a polyT tail. It can have a minimum length of 8-11nt.
  • at least a portion of the oligonucleotide is complementary to the human mautre miR-138 sequence or a fragment thereof.
  • Oligonucleotides targeting miR-138 can be designed in light of the sequence of miR-138, which is well known in the art, by routine technology.
  • the oligonucleotide described above for suppressing miR-138 can include non-naturally-occurring nucleobases, sugars, or covalent internucleoside linkages (backbones).
  • Such a modified oligonucleotide confers desirable properties such as enhanced cellular uptake, improved affinity to the target nucleic acid, and increased in vivo stability.
  • the oligonucleotide to be used in a method as described above has a modified backbone, including those that retain a phosphorus atom (see, e.g., U.S. Pat. Nos. 3,687,808 ; 4,469,863 ; 5,321,131 ; 5,399,676 ; and 5,625,050 ) and those that do not have a phosphorus atom (see, e.g., U.S. Pat. Nos. 5,034,506 ; 5,166,315 ; and 5,792,608 ).
  • Examples of phosphorus-containing modified backbones include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having 3'-5' linkages, or 2'-5' linkages.
  • Such backbones also include those having inverted polarity, i.e., 3' to 3', 5' to 5' or 2' to 2' linkage.
  • Modified backbones that do not include a phosphorus atom are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • Such backbones include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • the miR-138-targeting oligonucleotide described herein includes one or more substituted sugar moieties.
  • substituted sugar moieties can include one of the following groups at their 2' position: OH; F; O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl, and O-alkyl-O-alkyl.
  • the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • substituted sugar moieties include those having 2'-methoxyethoxy, 2'-dimethylaminooxyethoxy, and 2'-dimethylaminoethoxyethoxy. See Martin et al., Helv. Chim. Acta, 1995, 78, 486-504 .
  • the oligonucleotide described herein includes one or more modified native nucleobases (i.e., adenine, guanine, thymine, cytosine and uracil).
  • Modified nucleobases include those described in U.S. Pat. No. 3,687,808 , The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990 , Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613 , and Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, CRC Press, 1993 .
  • nucleobases are particularly useful for increasing the binding affinity of the oligonucleotide to miR-138.
  • These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines (e.g., 2-aminopropyl-adenine, 5-propynyluracil and 5-propynylcytosine). See Sanghvi, et al., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278 ).
  • a miR-138 targeting oligonucleotide can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition.
  • a pharmaceutically acceptable carrier is a carrier compatible with the active ingredient of the composition (and preferably, stabilizes the active ingredient) and not deleterious to the subject to be treated.
  • Suitable carriers include, but are not limited to, (a) salts formed with cations (e.g., sodium, potassium, ammonium, magnesium, calcium) and polyamines (e.g., spermine and spermidine); (b) acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid); (c) salts formed with organic acids (e.g., acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid); and (d) salt
  • Suitable carriers include microcrystalline cellulose, mannitol, glucose, defatted milk powder, polyvinylpyrrolidone, starch, and a combination thereof. See, e.g., Remington's Pharmaceutical Sciences, Edition 18, Mack Publishing Co., Easton, Pa (1995 ); and Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Tenth Edition, Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001 .
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration (e.g., intrathecal or intraventricular).
  • An injectable composition containing an oligonucleotide for targeting miR-138 may contain various carriers such as vegetable oils, dimethylactamide, dimethylormamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).
  • the oligonucleotide can be administered by the drip method, whereby a pharmaceutical formulation containing the oligonucleotide and a physiologically acceptable excipients is infused.
  • Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients.
  • Intramuscular preparations e.g., a sterile formulation of a suitable soluble salt form of peptide, can be dissolved and administered in a pharmaceutical excipient such as sterile water, 0.9% saline, or 5% glucose solution.
  • the oligonucleotide When oral administration is applied, it is preferred that the oligonucleotide includes at least one 2'-O-methoxyethyl modification.
  • a composition for oral administration can be any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.
  • carriers which are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried corn starch.
  • aqueous suspensions or emulsions When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.
  • a nasal aerosol or inhalation composition can be prepared according to techniques well known in the art of pharmaceutical formulation. The pharmaceutical composition described herein can also be administered in the form of suppositories for rectal administration.
  • the oligonucleotide can be conjugated with a chaperone agent.
  • conjugated means two entities are associated, preferably with sufficient affinity that the therapeutic benefit of the association between the two entities is realized. Conjugated includes covalent or noncovalent bonding as well as other forms of association, such as entrapment of one entity on or within the other, or of either or both entities on or within a third entity (e.g., a micelle).
  • the chaperone agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin, low-density lipoprotein, or globulin), carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipid. It can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.
  • a protein e.g., human serum albumin, low-density lipoprotein, or globulin
  • carbohydrate e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid
  • lipid e.g., a recombinant or synthetic molecule, such as a synthetic polymer, e.g.,
  • polyamino acids examples include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, and polyphosphazine.
  • PLL polylysine
  • poly L-aspartic acid poly L-glutamic acid
  • styrene-maleic acid anhydride copolymer examples include poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacryl
  • the chaperone agent is a micelle, liposome, nanoparticle, or microsphere, in which the oligonucleotide/interfering RNA is encapsulated.
  • Methods for preparing such a micelle, liposome, nanoparticle, or microsphere are well known in the art. See, e.g., U.S. Pat. Nos. 5,108,921 ; 5,354,844 ; 5,416,016 ; and 5,527,5285 .
  • the chaperone agent serves as a substrate for attachment of one or more of a fusogenic or condensing agent.
  • a fusogenic agent is responsive to the local pH. For instance, upon encountering the pH within an endosome, it can cause a physical change in its immediate environment, e.g., a change in osmotic properties which disrupts or increases the permeability of the endosome membrane, thereby facilitating release of the miR-138-targeting oligonucleotide into host cell's cytoplasm.
  • a preferred fusogenic agent changes charge, e.g., becomes protonated at a pH lower than a physiological range (e.g., at pH 4.5-6.5).
  • Fusogenic agents can be molecules containing an amino group capable of undergoing a change of charge (e.g., protonation) when exposed to a specific pH range.
  • Such fusogenic agents include polymers having polyamino chains (e.g., polyethyleneimine) and membrane disruptive agents (e.g., mellittin).
  • polymers having polyamino chains e.g., polyethyleneimine
  • membrane disruptive agents e.g., mellittin
  • Other examples include polyhistidine, polyimidazole, polypyridine, polypropyleneimine, and a polyacetal substance (e.g., a cationic polyacetal).
  • a condensing agent interacts with the oligonucleotide, causing it to condense (e.g., reduce the size of the oligonucleotide), thus protecting it against degradation.
  • the condensing agent includes a moiety (e.g., a charged moiety) that interacts with the oligonucleotide via, e.g., ionic interactions.
  • condensing agents include polylysine, spermine, spermidine, polyamine or quarternary salt thereof, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, and alpha helical peptide.
  • miR-138 is a reliable biomarker for GSCs, which play crucial roles in MG initiation, progression, and metastasis. Presence of GSCs in a subject is indicative of an increased risk for MG occurrence (including occurrence of grade-III anaplastic astrocytoma or grade-IV glioblastoma multiforme), progression, and/or metastasis.
  • a method for identifying GSCs in a sample suspected of containing GSCs is a method for identifying GSCs in a sample suspected of containing GSCs.
  • a sample e.g., a brain tumor biopsy sample
  • a conventional method such as quantitative real-time PCR and hybridization, can be performed to quantify the amount of miR-138 in the sample.
  • Oligonucleotide primers or probes complementary to a portion of miR-138 can be designed for amplifying miR-138 or hybridizing to this micro RNA. It was within the knowledge of a skilled person to design primers for amplification of miR-138.
  • Oligonucleotide probes specific to miR-138 are capable of hybridizing to this target miRNA under, e.g., stringent hybridization conditions.
  • stringent conditions refers to parameters known to those skilled in the art.
  • One example of stringent conditions is hybridization at 65 °C. in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin (BSA), 25 mM NaH 2 PO 4 (pH7), 0.5% SDS, 2 mM EDTA).
  • SSC 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS is sodium dodecylsulphate; and EDTA is ethylene diamine tetra acetic acid.
  • SSC 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS is sodium dodecylsulphate; and EDTA is ethylene diamine tetra acetic acid.
  • the miR-138 amount thus determined is compared with a predetermined standard.
  • An elevated level of miR-138 in the sample indicates presence of GSCs.
  • the predetermined standard represents the average amount of miR-138 in non-tumor cells, e.g., normal brain cells, which preferably are from subjects with matched age, sex, and/or ethnic background.
  • the predetermined standard is the level of miR-138 in neural stem cells.
  • miR-138 As also disclosed herein, high levels of miR-138 was observed in recurrent GBM patients as compared with healthy controls. Thus, the level of this microRNA is a prognostic biomarker indicating recurrence of GBM.
  • a biosample e.g., a brain tissue biopsy
  • the amount of miR-138 can be determined via any method known in the art.
  • the miR-138 level can be compared with a predetermined standard to determine whether the patient has GBM, and if so, the stage of the disease. More specifically, if the miR-138 level in the sample is higher than the predetermined standard, it indicates that the patient has a relapse of GBM and as a malignant tumor, indicates poor survival rates.
  • the predetermined standard represents the average amount of miR-138 in non-glioblastoma patients (e.g., healthy control subjects) or in patients first detected with MG.
  • the predetermined standard is obtained from patients with matched age, sex, and/or ethnic background relative to the MG patient whose disease stage needs to be determined.
  • biosamples such as those described above can be obtained from the patient at multiple time points and the amounts of miR-138 in the samples are measured via a routine method. If the amount of miR-138 increases over time, it indicates that MG is progressing in the patient.
  • biosamples can be obtained from a subject having MG before, during, or after a MG treatment.
  • the amounts of miR-138 in the samples can be determined by a routine method.
  • a pre-treatment sample and a posttreatment sample are obtained.
  • samples at multiple time intervals during a treatment can be obtained.
  • the miR-138 level remains unchanged or decreases after the treatment or along the course of the treatment (that is, the levels of miR-138 remain the same or are lower in samples taken at intervals later in the course of the treatment as compared to those in samples taken at intervals earlier in the course of the treatment), it indicates that the treatment is effective.
  • This method can be applied to a human MG patient to evaluate whether the patient is responsive to a MG treatment. It also can be applied to a laboratory animal used in developing new MG treatment.
  • GSC cell lines used in the study were derived from five malignant glioma patient samples NNI-1, NNI-4, NNI-8, NNI-11, and NNI-12. Briefly, the GSC cell lines were cultured in chemically defined serum-free selection growth medium consisting of 20 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF), 20 ng/ml human recombinant leukemia inhibitory factor (LIF), 5 ug/ml heparin, serum-free supplement (B27) in a 3:1 mix of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 Nutrient Mixture (F12).
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • LIF human recombinant leukemia inhibitory factor
  • B27 serum-free supplement
  • DMEM Dulbecco's modified Eagle's medium
  • F12 Ham's F-12 Nutrient Mixture
  • the cultures were incubated at 37 °C in a water-saturated atmosphere containing 5% CO 2 . Growth factors were replenished every other day. Tumorsphere cultures were expanded by mechanical trituration using a flame-drawn glass Pasteur pipette.
  • NSCs Normal human neural stem cells
  • bFGF basic fibroblast growth factor
  • EGF endothelial growth factor
  • B27 2% B27
  • Lenti-X 293T (Clontech) were cultured according to the supplier's instructions. Human MG specimens for in situ hybridization were obtained from the National University Hospital (NUH).
  • Spheres were harvested and dissociated into single cells. Further, single cells were plated into 96-well by manual picking. After plating, cells were observed and only wells with single cell were considered. The number of wells with tumor spheres was scored after 21 days.
  • RNAs from GSCs and NSCs was isolated using Exiqon kit following manufacturer's protocol. RNA was subjected to quality control analysis by nano-drop and Bioanalyzer to assess the concentration, integrity of the RNAs and the content of small RNAs. Further, the RNAs thus obtaiend was subjected to miRCURYTM LNA Array microRNA profiling (Exiqon) following manufacturer's protocol. Arrays were scanned in ozone free environment. Image analysis was performed to quantify the signals on the arrays. Technical quality assessment was performed based on results from spike in controls, flagging of spots, background intensity levels and signal intensity distribution. After background subtraction and normalization, differential gene expression analysis was performed on, e.g., log2(Hy3/Hy5) ratios including the top-50 miRNA (lowest p-value). A multiple ANOVA model was used to remove batch effects from replicate samples.
  • RNA 500 ng
  • Biotinylated RNA was isolated using Qiagen mini columns according to the manufacturer's instructions.
  • cRNA was hybridized on HumanWG-6 v3.0 array chips from Illumina (Illumina Inc., San Diego, CA), washing and scanning steps were performed according to the Illumina BeadStation 500X manual. The data was extracted, normalized, and analyzed using Illumina BeadStudio provided by the manufacturer.
  • Transcript signals that were below the limit of detection (LOD) at 99% confidence were defined as genes not expressed.
  • Microarray analysis was performed on PARTEK platform. Gene datasets specific for each group of cells were uploaded onto Ingenuity Pathway Analysis platform (Ingenuity Systems, Mountain View, CA) for analysis of biological process, molecular functions and pathways.
  • RNA-enriched total RNA was separated on 15% denaturating Urea-PAGE, transferred to positively charged nylon membrane (Ambion, Austin, TX), and cross linked at 120 mJ in UV Stratagene cross linker 2400.
  • MiRCURY LNA probes for miR-138 and U6, were end-labeled with T4 polynucleotide kinase followed by hybridization.
  • RNA polymerase III Stable expression of antimiRs were carried out using miRZipTM expression vector, a third generation HIV-based expression Lentiviral vector provided by System Biosciences, LLC.
  • This vector has a CMV-driven [GFP-T2A-Puromycin] reporter cassette and a H1 promoter upstream of either antigomiR-138 or a control non-targeting small RNA.
  • the hairpin consists of 23 nt sense sequence, a 11 nt short spacer (CTTCCTGTCAG; SEQ ID NO:3), an antisense sequence, 6 Ts (a transcription stop signal for RNA polymerase III).
  • the antigomiR sequence used to construct lentivirus against human mature-miR-138 was 5'-GAGCTGGTATTGTGAATCAAGCAGCTTCCTGTCAGCGGCCTGATTCA CAACACCAGCTTTTTT3' (SEQ ID NO:2).
  • the mature functional antigomiR-138 sequence is CGGCCTGATTCACAACACCAGCT (SEQ ID NO:1), which is complementary to the mature miR-138 sequence.
  • the H1 expression cassette of the vector provides constitutive and efficient RNA polymerase III-dependent transcription of antigomiR transcripts.
  • CMV promoter promotes high level of expression of both copGFP (fluorescent reporter) and puromycin-Nacetyl transferase (drug selectable marker) in the same vector for detection and selection of transduced cells.
  • synthetic single-stranded anti-miRNA or antigomiRs were introduced into cells to knock down miRNAs (i.e., decreasing miRNA levels) transiently.
  • Lenti-X 293T (cat# 632180, Clontech, Mountain View, CA) devoid of Tat in the packaging mix.
  • Super coiled DNA of constructs were prepared by using Qiagen maxi prep kits (cat # 1045091, Germantown, MD). The packaging constructs were used to transfect Lenti-X 293T cells as described in the protocol in Tiscomia et al., 2006.
  • Transducing Units determination and transductions were performed following the methods also described in Tiscomia et al., 2006. Transfections were performed in antibiotic free medium using EFFECTENE transfection reagent (cat # 301427, Qiagen, Germantown, MD) reagent as per manufacturer protocol. Supernatant was collected 2nd and 3rd day post transfection. Supernatant is pooled and filtered through a 0.45 ⁇ m HV Durapore membrane (Millipore # SCHVU01RE) Filtrate is subjected to centrifugation at 25,000 rpm ( ⁇ 82,000 g) for 2h at 4 °C. The visible pellet was re-suspended in cold 1X HBSS (cat # 9269, Sigma-Aldrich, St. Louis, MO, USA) and stored at -80 °C or used for transduction.
  • EFFECTENE transfection reagent catalog # 301427, Qiagen, Germantown, MD
  • Supernatant was collected 2nd and
  • Transducing Units were determined as per the protocol described also in Tiscornia et al., 2006. Lenti viral titrations were performed in HEK 293T cells. Briefly, HEK293T cells were plated at density of 4X10 4 /well in 24-well plate. Next day, cells were transduced with serially diluted lentiviruses in the presence of polybrene 8 ug/ ml. Three days of post transduction, percentage GFP positive cells were measured using FACS analysis and number of transducing particles was determined.
  • single cell suspension was made by ACCUTASE (Invitrogen) treatment and resuspended in complete medium containing 0.3% noble agar (Difco).
  • a total of 2x10 3 cells were seeded in each well of 6 well plates (Nunc) containing 1% noble agar. Cells were cultured for 2-3 weeks by supplementing fresh medium with growth factors and, once in every three days. Lentiviral transduced cells were grown in the presence of 1 ug/ ml puromycin.
  • Cells infected with lentivirus for 48 h were plated at a density of 5,000 cells/well in 96-well plates in at least triplicate for growth curve analysis. Live cells were determined at the indicated number of days after plating using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega).
  • Luciferase reporter constructs containing the 3'UTR of BLCAP, CASP3 and MXD1 were purchased from Genecoepia (Rockville, MD). Total of 50 ng of wild-type or mutated reporter plasmids were cotransfected with 250 ng of pCDH-miR-138 or a negative control vector into HEK293T cells using EFFECTENE following the manufacturer's protocol (Qiagen). Firefly and Renilla luciferase activities were measured at 48 hours of post-transfection using Dual-Luciferase Reporter System (Promega) according to the manufacturer's instructions. The firefly luminescence was normalized to the Renilla luminescence as an internal control for determining transfection efficiency.
  • miR-138 binding site was mutated using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene), in accordance with the manufacturer's instructions.
  • the miR-138 binding site accagc was substituted with cttgat, gaagta and gcagaa in BLCAP, MXD1, and CASP3 respectively.
  • GSCs were infected with lentivirus for 2 days.
  • Cells were plated at a density of 5000 cells/well in 96-well lates in at least triplicate.
  • Caspase 3/7 activity was measured by commercially available Caspase-Glo 3/7 assay kit (Promega). Relative luciferase units were calculated based on the number of viable cells, using the CellTiter-Glo Luminescent Cell Viability assay as described above.
  • GSCs plated in six-well plates at 100,000 cells per well were infected with Lentiviruses for 48 hours.
  • cells were labeled with 20 ⁇ M EdU for 5 hours prior to harvesting, and processed using the Click-iT EdU Alexa Fluor 647 Flow Cytometry assay kit (Invitrogen), and cell nuclei were counter stained with 25 ⁇ g/ml propidium iodide (Sigma-Aldrich). Samples were subject to EdU incorporation analysis on a BD FACS caliber (Becton Dickinson). Data were analyzed using the WIN-MDI 2.9 software. To detect apoptotic cells, Annexin V-FITC staining was performed with the Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen) following manufacturer's protocol.
  • BD Pharmingen Annexin V-FITC staining was performed with the Annexin V-FITC Apoptosis Detection Kit (BD Pharm
  • Lentiviral transduced spheres were grown for indicated time points and collected by spinning at 900 x g. Spheres were washed twice with PBS and paraffin embedded sections were made and attached on 0.01% polylysine-coated slides, fixed with 4% methanol-free formaldehyde solution, bleached with 6% H 2 O 2 and fluorescence stained by following the procedure of the DeadEnd fluorometric TUNEL system (Promega). Cell nuclei were counter stained with DAPI.
  • Equal amount of cell lysate were resolved by SDS-PAGE, transferred to PolyVinylidene difluoride membranes (Millipore), and detected using SuperSignal West Pico (Pierce Biotechnology). All blots were stripped and reprobed with ⁇ -actin antibodies (Abcam) as loading control.
  • primary antibodies used were: anti-PARP (Abcam), anti-LASP1 (Abcam), anti-BTG2 (Abcam); anti- OLIG1 (Abcam), anti-GADD45 ⁇ (Santa Cruz), anti-EFNA1 (Santa Cruz), anti-CYCLIN D1 (Santa Cruz), anti-CDKN1A (Santa Cruz), anti-AURKA (Cell signaling), anti-phospho-Histone3 (Millipore), anti-Pannexin 2 (Santa Cruz), anti-TXNIP (Santa Cruz), Secondary antibodies used were anti-rabbit HRP (Santa Cruz) and anti-mouse HRP (Jackson Laboratory).
  • Luciferase reporter constructs containing the 3'UTR of BLCAP, CASP3 and MXD1 were purchased from Genecoepia (Rockville, MD). Total of 50 ng of wild-type or mutated reporter plasmids were cotransfected with 250 ng of pCDH-miR-138 or negative control vector into HEK293T cells using Effectene as per manufactures protocol (Qiagen). Firefly and Renilla luciferase activities were measured at 48 hours of post-transfection using Dual-Luciferase Reporter System (Promega) according to the manufacturer's instructions. The firefly luminescence was normalized to the Renilla luminescence as an internal control for transfection efficiency. The experiments were performed in triplicates.
  • miR-138 binding site was mutated using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene), in accordance with the manufacturer's instructions.
  • the miR-138 binding site accagc was substituted with cttgat, gaagta and gcagaa in BLCAP, MXD1 and CASP3 respectively.
  • the DIG labeled LNA probes were detected using Alkaline phosphatase conjugated anti-DIG antibody and BM Purple substrate (Roche) at 30°C. Color development was continued until the scramble control LNA probe treated sections show non-specific staining. Slides were dehydrated, passed through Xylene bath and mounted with DPX-neutral mounting medium (Sigma). Images were acquired using Bright field microscope.
  • GSCs Luciferase stables were established by selecting for puromycin(1 ⁇ g/ml) after transducing with Lentivirus expressing Luciferase under human PGK promoter (Addgene # w543-1) (Campeau et al. 2009).
  • GSCs expressing firefly luciferase bearing scramble-control or antimiR-138 were intracranially injected into the right forebrains of NOD-SCID mice, and the Xenogen system was used for imaging. Mice were given an intraperitoneal dose of 150 mg/kg of D-luciferin and anesthetized anesthetized in a closed box filled with oxygen and 2-3% isoflurane.
  • mice were imaged after 15 minutes of D-luciferin dosage using the IVIS spectrum Imaging System (Xenogen). Quantification was based on total flux (photons/sec) of emitted light as a measure of the relative number of viable cells. Bioluminescence signals were analyzed using Living Image software (IVIS living image v3.0).
  • GSCs are multipotent and tumorigenic
  • GSCs were derived from tumor specimens obtained from five malignant glioma patients NNI-1, NNI-4, NNI-8, NNI-11 and NNI-12 (see Chong et al., 2009 and Foong et al., 2011) were found to retain their properties for limited passages. A comprehensive validation to test self-renewal properties in vitro and tumorigenic properties in an immune-compromised host was performed on these cell lines. GSCs were first characterized on the basis of functional criteria in comparison with normal human neural stem cell (NSCs). The NSCs used in this study were obtained from Lonza (whole brain-derived), or from specific regions of the human foetal brain (either the frontal cortex or the sub-ventricular zone).
  • GSCs In an undifferentiated state, in culture, GSCs form tumorspheres that resemble NSCs derived neurospheres ( Figure 1A ). GSCs, like NSCs, express CD133 (Prominin-1) and Nestin. These markers were found to be down-regulated on differentiation ( Figure 1B ). To examine the self-renewal capacity of GSCs we performed single cell (clonal) sphere-formation assays (Reynolds, et.al., 2005). When individual spheres from primary culture after enzymatic dissociation were plated as a single cells per well in a 96 well plate and the number of wells with a single cell were scored, 15% of wells formed tumorspheres.
  • GSCs like NSCs, were found to be multipotent and differentiated into neurons, astrocytes and oligodendrocytes ( Figure 1D ). However, unlike NSCs, the GSCs were found to be tumorigenic and the first appearance of well-defined tumor masses were observed at 12 weeks after sub-cutaneous injection of GSCs in NOD-SCID mice. Further, GSC-derived xenograft tumor sections were subjected to H&E staining. This histo-pathological analysis of GSC-derived tumors demonstrated a characteristic glioblastoma tissue pattern, e.g., the presence of large areas of necrosis surrounded by highly packed tumor cells.
  • the miRNA expression profiles of GSCs were compared with those of NSCs, using miRNA profiling analysis as described in the Method section above. Differentially expressed miRNAs were identified using ANOVA analysis on normalized microarray data. The scatter plot of the expression data reveals that most of the transcripts were within the 2-fold cut off band, suggesting that the overall miRNA expression pattern in NSCs is very similar to that of GSCs. Among the few differentially expressed transcripts, miR-138 emerged as the most consistent, differentially expressed miRNA. Fig. 2A . GSCs expressed elevated levels of miR-138 compared to the very low or no expression in NSCs.
  • miR-138 has two putative precursors pre-miR-138-1 and pre-miR-138-2, and the synthesis of mature miR-138 is regulated by stringent transcriptional and post-transcriptional control mechanisms.
  • the expression levels of precursor transcripts in GSCs and NSCs were examined. Elevated levels of pre-miR-138-2 were detected only in GSCs, not in NSCs ( Figure 2D ), suggesting that miR-138 is under tight transcriptional control in NSCs.
  • the expression levels of miR-138 were then analyzed when GSCs were subjected to differentiation. The results thus obtained show clear down-regulation of miR-138 in differentiated GSCs.
  • Figure 2E The results thus obtained show clear down-regulation of miR-138 in differentiated GSCs.
  • miR-138 is differentially expressed in GSCs and is therefore a reliable biomarker for identifying GSCs, the presence of which is indicative of an increased risk for MG initiation, progression, and metastasis.
  • lentiviral-based antagomirs were used to obtain stable loss-of-function phenotypes (Krutzfeldt et al., 2005 and Scherr et al., 2007).
  • a non-targeting-scrambled control (a scramble control) was used.
  • Sequence-specific inhibition of miRNA-138 by a lentiviral vectors encoding an anti-miRNA-138-antagomirs (antimiR-138) affected growth and survival of GSCs ( Figure 3A lower panel) and this is apparent by a decrease in size of tumorspheres compared to the scramble-control ( Figure 3A and 3B ).
  • miR -138 plays a pro-survival role in GSCs
  • TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling
  • miR-138 promotes tumorigenesis and is an essential pro-survival oncomiR for GSCs:
  • mice To evaluate whether disruption of miR-138 function ablates tumor formation in immunocompromised mice, equal number of viable cells infected with antimiR-138 or the scramble-control were transplanted sub-cutaneously or intracranially in 14 immune-compromised mice. Ten of twelve mice transplanted with GSCs transduced with the scramble developed tumors whereas none of the twelve mice transplanted with GSCs transduced with antimiR-138 GSCs develop tumors. The xenograft tumor sections display GFP and GFAP positive cells confirming that the GFP positive GSCs are tumorigenic.
  • ISH In-situ -hybridization
  • luciferase-expressing GSCs transfected with antimiR-138 were intracranially implanted into the forebrains of immune-compromised mice.
  • An equal number of luciferase-expressing GSCs transfected with the non-targeting-scramble RNA was used as a control.
  • the viability of the engrafted GSCs was measured by tracking the bioluminescence after implantation via real-time imaging at various time points as indicated in Figure 5A .
  • mice transplanted with the antimiR-138-expressing GSCs did not show neurological symptoms 18 months after the transplantation, while the percentage of neurological symptom-free mice transplanted with GSCs transfected with the scramble control decreased over time.
  • Figure 5C A Kaplan-Meier survival curve also demonstrated increased survival rates in mice transplanted with GSCs transduced with antimiR-138; mice transplanted with GSCs transduced with the scramble control developed tumors and showed a high percentage of mortality. All these results demonstrate that miR-138 positive GSCs are tumorigenic and functional knockdown of this pro-survival oncomiR leads to apoptotic death of GSCs and impedes tumor formation.
  • miR - 138 targets tumor suppressor genes, pro-apoptotic gene, and proliferation inhibitor genes
  • targets of miR-138 were identified by comparing gene expression profiles from GSCs transduced with either antimiR-138 or the scramble control.
  • Figure 6A displays several differentially expressed genes.
  • MYC, BCL2, CASC4, CCND1, HIF1a, LASP1, OLIG1, AURKA, and CCNA2 were found to be down-regulated in GSCs expressing antigomiR-138 as compared with GSCs expressing the scramble control; and RARA, GADD45A, CASP3, MXD1, PANX2, COL4A1, TXNIP, EFNA1, TUSC2, BLCAP, and BTG2 were found to be up-regulated in GSCs expressing antigomiR-138 as compared with GSCs expressing the scramble control.
  • MXD1 MAX dimerization protein 1
  • TUSC2 Tumor suppressor candidate 2
  • BLCAP Bladder cancer-associated protein
  • CRISPR3 Cysteine-aspartic acid protease-3
  • BCG2 B-cell translocation gene 2
  • EFNA1 Ephrin-A1
  • Pannexin2 Pannexin2
  • TXNIP thioredoxin interacting protein
  • Targetscan v5.1 database and correlation of the predicted targets with microarray data revealed potential direct targets of miR-138.
  • miR-138 include CASP3, BLCAP and MXD1, each with one conserved binding site for miR-138.
  • Chimeric 3'-UTR luciferase reporter assays were performed to confirm the ability of miR-138 to negatively regulate CASP3, MXD1 and BLCAP expression, Expression of miR-138 resulted in significant down-regulation of CASP3, MXD1, or BLCAP 3'-UTR luciferase reporter activity, whereas the reporter vector with a mutation in the miR-138 binding site was not affected confirming that CASP3, MXD1 and BLCAP are direct targets of miR-138.
  • Figure. 6D .
  • miR-138 indirectly activates a plethora of genes involved in tumorigenesis via targeting tumor suppressors, inhibitors of proliferation, and transcriptional repressors, miR-138.
  • Down-regulation of cyclin D1 (CCND1), cyclin A2 (CCNA2), aurora kinase A (AURKA), and proto-oncogene c-Myc (cMYC) by miR-138 inhibition confirms this conclusion.
  • Figure 6A and 6C .
  • miR-138 As a confirmation of clinical significance, and to confirm what we observed above is not a culture artifact, we screened for miR-138 on patient samples. Expression levels of miR-138 in patient tumor specimens were examined by in situ hybridization, using a miR-138-specific probe on patient-derived GBM tumor sections and normal human brain frontal lobe sections. Positive signals were detected in patient-derived tumor sections stained with the miR-138 specific-probe but not in normal human brain sections stained with the same probe. This result confirms the expression of miR-138 in GBM specimens.
  • Figure 8B ISH was performed on 25 patient samples and the results suggested a discrete expression pattern for miR-138 in GBMs.

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Claims (15)

  1. Composition pharmaceutique destinée à être utilisée dans le traitement d'un glioblastome multiforme, comprenant un oligonucléotide ciblant miR-138.
  2. Composition pharmaceutique destinée à être utilisée selon la revendication 1, la quantité de l'oligonucléotide étant efficace pour la suppression de la prolifération de cellules souches de gliome ou l'induction de l'apoptose dans des cellules souches de gliome.
  3. Composition pharmaceutique destinée à être utilisée selon la revendication 1 ou 2, l'oligonucléotide étant un oligonucléotide en épingle à cheveux,
    éventuellement un ou plusieurs des nucléotides dans l'oligonucléotide étant modifiés par un groupe 2'-O-méthoxy, un groupe 2'-O-méthoxyéthyle ou un groupe phosphorothioate,
    éventuellement l'oligonucléotide comprenant la séquence nucléotidique de 5'-CGGCCTGATTCACAACACCAGCT-3' (SEQ ID n° : 1),
    éventuellement l'oligonucléotide comprenant la séquence nucléotidique de 5'-GAGCTGGTATTGTGAATCAAGCAGCTTCCTGTCAGCGGCCTGATTCACAACACCA GCTTTTTT-3' (SEQ ID n° : 2).
  4. Oligonucléotide ciblant miR-138 pour le traitement d'un glioblastome multiforme chez un sujet en ayant besoin.
  5. Oligonucléotide destiné à être utilisé selon la revendication 4, l'oligonucléotide étant efficace pour la suppression de la prolifération de cellules souches de gliome,
    éventuellement l'oligonucléotide étant efficace pour l'induction de l'apoptose dans des cellules souches de gliome,
    éventuellement le sujet étant un sujet humain souffrant de, soupçonné d'avoir ou risquant de développer un glioblastome multiforme,
    éventuellement l'oligonucléotide étant un oligonucléotide en épingle à cheveux,
    éventuellement un ou plusieurs des nucléotides dans l'oligonucléotide étant modifiés par un groupe 2'-O-méthoxy, un groupe 2'-O-méthoxyéthyle ou un groupe phosphorothioate,
    éventuellement l'oligonucléotide comprenant la séquence nucléotidique de 5'-CGGCCTGATTCACAACACCAGCT-3' (SEQ ID n° : 1),
    éventuellement l'oligonucléotide comprenant la séquence nucléotidique de 5'-GAGCTGGTATTGTGAATCAAGCAGCTTCCTGTCAGCGGCCTGATTCACAACACCA GCTTTTTT-3' (SEQ ID n° : 2).
  6. Oligonucléotide ciblant miR-138 destiné à être utilisé dans l'inhibition de la croissance de cellules souches de gliome chez un sujet en ayant besoin.
  7. Oligonucléotide destiné à être utilisé selon la revendication 6, dans lequel
    éventuellement le sujet est un sujet humain souffrant de, soupçonné d'avoir ou risquant de développer un glioblastome multiforme,
    éventuellement l'oligonucléotide est efficace pour la suppression de la prolifération de cellules souches de gliome,
    éventuellement l'oligonucléotide est efficace pour l'induction de l'apoptose dans des cellules souches de gliome,
    éventuellement l'oligonucléotide est un oligonucléotide en épingle à cheveux, éventuellement un ou plusieurs des nucléotides dans l'oligonucléotide sont modifiés par un groupe 2'-O-méthoxy, un groupe 2'-O-méthoxyéthyle ou un groupe phosphorothioate,
    éventuellement l'oligonucléotide comprend la séquence nucléotidique de 5'-CGGCCTGATTCACAACACCAGCT-3' (SEQ ID n° : 1),
    éventuellement l'oligonucléotide comprend la séquence nucléotidique de 5'-GAGCTGGTATTGTGAATCAAGCAGCTTCCTGTCAGCGGCCTGATTCACAACACCA GCTTTTTT-3' (SEQ ID n° : 2).
  8. Oligonucléotide ciblant miR-138 pour prolonger la survie d'un sujet souffrant d'un gliome malin.
  9. Oligonucléotide destiné à être utilisé selon la revendication 8, la quantité de l'oligonucléotide étant efficace pour la suppression de la prolifération de cellules souches de gliome,
    éventuellement l'oligonucléotide étant efficace pour l'induction de l'apoptose dans des cellules souches de gliome,
    éventuellement le sujet étant un patient humain atteint d'un gliome malin,
    éventuellement le patient humain ayant subi ou étant en cours d'un ou plusieurs traitements supplémentaires de gliome malin,
    éventuellement ledit un ou plusieurs traitements supplémentaires de gliome malin étant une intervention chirurgicale, une radiothérapie et/ou une chimiothérapie,
    éventuellement l'oligonucléotide étant un oligonucléotide en épingle à cheveux, éventuellement un ou plusieurs des nucléotides dans l'oligonucléotide étant modifiés par un groupe 2'-O-méthoxy, un groupe 2'-O-méthoxyéthyle ou un groupe phosphorothioate,
    éventuellement l'oligonucléotide comprenant la séquence nucléotidique de 5'-CGGCCTGATTCACAACACCAGCT-3' (SEQ ID n°: 1),
    éventuellement l'oligonucléotide comprenant la séquence nucléotidique de 5'-GAGCTGGTATTGTGAATCAAGCAGCTTCCTGTCAGCGGCCTGATTCACAACACCA GCTTTTTT-3' (SEQ ID n° : 2).
  10. Méthode permettant de surveiller la progression d'une tumeur chez un patient atteint d'un gliome malin, comprenant :
    la quantification des taux de miR-138 dans des échantillons contenant des cellules souches de gliome provenant du patient obtenus à de multiples moments dans le temps, et
    l'évaluation pour savoir si le gliome malin progresse chez le sujet, une augmentation des taux de miR-138 au cours du temps indiquant la progression du gliome malin.
  11. Méthode selon la revendication 10, le patient atteint d'un gliome malin étant un patient humain ou un animal de laboratoire non humain, éventuellement, l'étape de quantification étant effectuée par PCR quantitative en temps réel ou par hybridation in situ.
  12. Méthode d'évaluation de l'efficacité d'un traitement de gliome malin chez un patient atteint d'un gliome malin, la méthode comprenant :
    la quantification des taux de miR-138 dans des échantillons contenant des cellules tumorales provenant du patient avant, pendant et/ou après le traitement, et
    l'évaluation de l'efficacité du traitement sur la base des taux de miR-138,
    aucun changement ou une diminution des taux de miR-138 après le traitement ou au cours du traitement indiquant que le traitement est efficace.
  13. Méthode selon la revendication 12, les échantillons provenant du patient avant et après le traitement,
    éventuellement les échantillons provenant du patient à de multiples intervalles pendant le traitement,
    éventuellement le patient atteint d'un gliome malin étant un patient humain ou un animal de laboratoire non humain,
    éventuellement l'étape de quantification étant effectuée par PCR quantitative en temps réel ou par hybridation in situ.
  14. Méthode d'identification de cellules souches de gliome, comprenant :
    la quantification du taux de miR-138 dans un échantillon soupçonné de contenir des cellules souches de gliome, et
    la détermination pour savoir si l'échantillon contient des cellules souches de gliome sur la base du taux de miR-138, un taux élevé de miR-138 dans l'échantillon par rapport à un étalon prédéfini indiquant la présence de cellules souches de gliome dans l'échantillon.
  15. Méthode selon la revendication 14, l'étalon prédéfini étant le taux de miR-138 dans des cellules non tumorales,
    éventuellement les cellules non tumorales étant des cellules souches neurales, éventuellement l'échantillon provenant d'un sujet humain,
    éventuellement comprenant en outre l'évaluation pour savoir si le sujet humain est soupçonné d'avoir ou risque de développer un gliome malin, la présence de cellules souches de gliome dans l'échantillon indiquant que le sujet humain est soupçonné d'avoir ou risque de développer un gliome malin,
    éventuellement le sujet humain étant un patient atteint d'un gliome malin et la méthode comprenant en outre l'évaluation du risque de progression de la tumeur ou de métastases tumorales chez le patient, la présence de cellules souches de gliome dans l'échantillon étant indicatrice d'un risque accru de progression du gliome malin ou de métastases,
    éventuellement l'étape de quantification étant effectuée par PCR quantitative en temps réel ou par hybridation in situ.
EP11849941.7A 2010-12-20 2011-12-20 Ciblage de cellules souches de gliome par une inhibition fonctionnelle spécifique de l'oncogène mir-138 pro-survie Active EP2654801B1 (fr)

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EP2654801A4 (fr) 2015-06-17
SG191174A1 (en) 2013-07-31
US9308217B2 (en) 2016-04-12

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